Acute coronary thrombosis results in myocardial infarction (Ml)with irreversible loss of functional myocardium unless reperfusion therapy is rapidly instituted. Many patients do not present or are not amenable to prompt reperfusion, and in this large patient population new therapeutic approaches are needed. Initial studies of cell-based therapies post-Mi in animal models and in patients have provided encouraging results, but the ideal donor cell population has not been determined. Preliminary data show that transplantation of undifferentiated mouse embryonic stem cells (ESCs) post-Mi, resulted in myocardial repair in a mouse Ml model; however, major mechanistic questions and roadblocks remain such as the risk of tumor formation. We hypothesize that cell preparations derived from ESCs that are committed to mesodermal or cardiac lineages can repair the myocardium with minimal risk of tumorigenesis. Secondly, we hypothesize that the optimal benefit from transplanted ESC-derived cells is the result of a short-term reduction in infarct size via paracrine effects and long-term regeneration of myocardium resulting from the proliferation and differentiation of transplanted cells in the absence of significant fusion. The long-term goal of this research is to develop cellular therapies using ESCs to repair the myocardium following Ml by providing essential preclinical studies of efficacy and safety. We propose to: 1) identify and characterize ESC-derived donor cells optimal for myocardial repair with minimal risk of tumorigenesis using cell surface markers and genetic cell-type specific reporters to isolate mesodermal progenitor cells and embryonic ventricular myocytes; 2) develop strategies to condition donor cells for improved survival in the setting of ischemic and oxidative stress typical of the post-Mi heart employing in vitro models of ischemia and reperfusion; and 3) determine the effect of transplantation of ESCs and derivatives post-Mi evaluating both short-term (infarct size, cell survival, cell proliferation) and long-term (LV structure and function by echocardiography and histology, tumor surveillance with microCT and pathology, cell fate/fusion by co- immunolabeling and Cre/lox donor/recipient techniques, and regenerated myocytes phenotype using cellular electrophysiology) outcomes. Overall, these studies will provide new insights into cell-based therapies in the post-Mi setting and bring approaches using human ESCs closer to clinical application.